Myocardial stunning and infarction are a major cause of morbidity and mortality after heart surgery, heart transplantation, and noninvasive myocardial revascularization. The long-term goal of the applicant is to develop new therapeutic strategies to improve myocardial tolerance to ischemia by targeting the heart's endogenous defense mechanisms against oxidative stress. This competing continuation application focuses on the beneficial effects of the endogenous compounds adenosine and pyruvate. Significant new findings indicate that adenosine receptor activation and pyruvate may both reduce reactive oxygen species (ROS) formation and increase antioxidant defenses in the ischemic-reperfused heart. The specific aims of the proposed research are to determine: 1) the role, if any, myocyte adenosine A3 and A2, receptors play in mediating adenosine-induced myocardial protection; 2) the mechanism(s) whereby adenosine receptor activation and pyruvate reduce cellular and subcellular ROS formation and oxidative stress; and, 3) the role adenosine and pyruvate play in enhancing endogenous antioxidant defense mechanisms. Studies will be performed in a porcine in vivo regional ischemia preparation, isolated perfused rat hearts, and rat isolated ventricu1ar myocytes. In myocytes ROS will be measured with the fluorescent probe dichlorofluorescein diacetate, and mitochondrial oxidative stress will be estimated by monitoring mitochondrial flavoprotein autofluorescence. The cardiac microdialysis technique will be used to measure interstitial fluid levels of ROS and the lipid peroxidation product malonaldehyde in the in vivo porcine studies. Oxidative stress and cell death (apoptosis and necrosis) will be measured in myocytes submitted to simulated ischemia reperfusion preparation will be used t o estimate interstitial fluid (ISF) levels of hydroxyl radical and. Antioxidant levels will be determined by measuring reduced glutathione, oxidized glutathione, the NADPH/NADP ratio, and expression of the antioxidant/anti-apoptotic protein Bcl-2 and the pro-oxidant/pro-apoptotic protein Bax. The effects of adenosine receptor agonists and pyruvate, alone and in combination, on oxidative stress and antioxidant status will be correlated with myocyte twitch amplitude and the extent of apoptotic and necrotic cell death following hypoxia-reoxygenation and simulated ischemia-reperfusion. The levels of ROS, malonaldehyde, and antioxidants in porcine myocardium will be correlated with load insensitive measurements of regional contractility and apoptotic and necrotic cell death. The proposed experiments are designed to elucidate the cellular and subcellular mechanisms of the salutary effects of adenosine receptor activation and pyruvate with the intent to further enhance their efficacy. The findings should lead to the development of new therapeutic strategies to improve hemodynamic performance and survival in patients with ischemic heart disease.